Long interspersed elements type 1 (L1s) are the only active autonomous transposable elements in our genome. L1s replicate themselves but also mobilize short interspersed elements and generate processed pseudogenes. This process is inherently mutagenic and accounts for over 0.1% of sporadic human diseases for which the underlying genetic alteration has been mapped. In addition, elevated L1 activity has been extensively documented during critical developmental stages (e.g., embryogenesis, neurogenesis, gametogenesis, and aging) and various disease processes (e.g., autoimmunity, genotoxic exposure, neurodevelopmental and neurodegenerative disorders, and tumorigenesis). However, the role of L1 retrotransposition during these developmental and disease processes remains poorly defined due to the lack of suitable mouse models. Existing mouse models utilize human L1-based transgenes, which are unlikely to be correctly regulated by endogenous mouse controlling mechanisms, especially when epigenetic mechanisms, such as small RNAs and DNA methylation, are involved. Additionally, the standard transgenesis approach has been used to make these mice and the resulting tandem arrayed transgenes are a target for repeat-induced gene silencing, confounding studies of the transcriptional regulation of these elements. Therefore, the objective of this proposal is to generate novel mouse models for L1 retrotransposons that can be used to interrogate the functional impact of L1 regulation and deregulation in vivo. The new transgenes will feature native mouse L1 elements, including the native promoter and two open reading frames. In addition, both L1 proteins will be epitope tagged to facilitate specific tracking of transgene expression and subcellular localization at the protein level; the transgenes will be marked by recently developed luciferase-based retrotransposition reporter cassettes; and the transgenes will be introduced into the mouse genome via targeted transgenesis to ensure single, dispersed copies. As a proof of principle, the timing of L1 expression and retrotransposition during male germ cell development will be characterized using these new transgenic mouse models. Similar analyses will be performed in a piRNA pathway mutant to evaluate the role of prepachytene piRNAs in controlling insertional mutagenesis by retrotransposons. The proposed project will be performed at one of the world's largest reproductive biology centers, which will allow us to integrate expertise from the fields of transposon biology and germ cell biology. In addition to facilitating our studies, the mouse models derived from this project will have broader applicability, e.g., when combined with other genetically manipulated disease models, they will be useful in elucidating the role of L1 activation under genetic, epigenetic and environmental perturbations, including areas of research sponsored by NCI, NIA, NIAAA, NICHD, NIDA, NIEHS, NIGMS, NIMH and NINDS.